Proteolysis of each of the complement proteins C3-C5 gives rise to aminoterminal cationic fragments with signalling molecules called anaphylatoxins. The most potent of these, C5a, elicits the broadest responses. Considering the components of the inflammatory response as margination and infiltration of leukocytes, release of granule-bound proteolytic enzymes, production of activated oxygen and nitrogen-derived radicals, changes in blood flow and capillary leakage, along with the ability to contract smooth muscle, the C5a molecule is the “complete” pro-inflammatory mediator. At sub-nanomolar to nanomolar levels, the C5a molecule elicits chemotaxis of all myeloid lineages (neutrophils, eosinophils and basophils, macrophages and monocytes), and causes vascular permeability which is markedly potentiated by prostaglandins and circulating leukocytes. Higher nanomolar concentrations elicit degranulation and activation of NADPH oxidase. This breadth of bioactivity contrasts with other inflammatory mediators. C5a has been implicated in the pathogenesis of rheumatoid arthritis, psoriasis, sepsis, reperfusion injury, and adult respiratory distress syndrome.
The activities of C5a are mediated by the binding of the C5a to its receptor (C5aR). C5aR belongs to the family of seven transmembrane G-protein-coupled receptors. C5aR is a high affinity receptor for C5a, with a Kd of ˜1 nM, and is located on a number of different cell types including leukocytes. The number of receptors per cell is extremely high, up to 200,000 sites per leukocyte. Biological activation of the receptor occurs over the range that saturates binding.
C5aR comprises an extended N-terminal extracellular domain. This large N-terminal domain is typical of G-protein coupled receptors which bind peptides including the IL-8 and fMet-Leu-Phe (FMLP) receptor families. The C5aR structure conforms to the seven transmembrane receptor family, with the extracellular N-terminus being followed by seven transmembrane helices connected by interhelical domains alternating as intracellular and extracellular loops, and ending with an intracellular C-terminal domain.
Agonists of C5aR are useful for therapeutic purposes, for example, in defence against bacterial infection, to stimulate immunoregulatory effects of C5a, and to treat cancers, immunodeficiency diseases and severe infections.
Antagonists of C5aR are also useful therapeutic agents, for example for treating inflammatory diseases and autoimmune disorders. For example, antagonists of C5aR are useful in the treatment of asthma, bronchial allergy, chronic inflammation, systemic lupus erythematosis, vasculitis, rheumatoid arthritis, osteoarthritis, gout, some auto-allergic diseases, transplant rejection, inflammatory bowel disease (for example, ulcerative colitis), in certain shock states, myocardial infarction, and post-viral encephalopathies. To this end, C5aR peptide antagonists and anti-C5a receptor antibodies have been previously described. For example, WO95/00164 describes antibodies directed against an N-terminal peptide (residues 9-29) of the C5a receptor.
Currently, alternative and/or improved C5aR antagonists and antagonists are desirable, as are improved methods of screening for C5aR antagonists and antagonists.
In vitro screening methods for detecting of C5aR agonists/antagonists are known in the art. For example, chemotaxis assays can be used to assess the ability of an antibody or functional fragment thereof to block binding of a ligand to C5aR and/or inhibit function associated with binding of the ligand to the receptor. These assays are based on the functional migration of cells in vitro induced by a compound. Chemotaxis can be assessed by any suitable means, such as in an assay utilizing a 96-well chemotaxis plate, or using other art-recognized methods for assessing chemotaxis. For example, the use of an in vitro transendothelial chemotaxis assay is described by Springer et al. (Springer et al., WO 94/20142, published Sep. 15, 1994; see also Berman et al., Immunol. Invest. 17: 625-677 (1988)). Migration across endothelium into collagen gels has also been described (Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991)).
A critical requirement in the drug discovery process is the demonstration in relevant animal models that new therapeutic agents identified by in vitro screening methods are safe and effective. Moreover, it is often desirable to compare the in vivo efficacy of numerous agents to select for desirable properties including pharmacokinetic properties, efficacy in affecting disease outcome and lack of adverse side effects. One of the main obstacles to drug development is moving drug candidates from in vitro assays to demonstration of in vivo efficacy. Often this is because many drug candidates are species-specific. For instance, an antagonist developed to a human chemoattractant receptor, such as C5aR, might antagonise only human C5aR and not C5aR from mouse, rabbit or even higher primates. The inability of many drugs candidates to “work” across species is a major reason for attrition in the preclinical phase.
Improved screening methods that allow identification and/or testing of human C5aR agonists, inverse agonists and antagonists in an in vivo environment are therefore desirable.